This application note describes a method for identifying oxidation sites on a monoclonal antibody (mAb) using an Agilent 1260 Infinity HPLC-Chip/MS system coupled to an Agilent Accurate-Mass 6520 Quadrupole Time-of-Flight (Q-TOF) LC/MS. A monoclonal antibody was oxidized by treatment with hydrogen peroxide to stimulate potential oxidative modifications that may occur during the manufacturing process. The accurate mass measurement of the 6520 Q-TOF LC/MS combined with Agilent MassHunter BioConfirm software facilitated easy identification of the oxidation sites. Peptides containing amino acid residues such as methionine and tryptophan, which are sensitive to oxidation, were easily identified using this approach.
Monoclonal antibodies can undergo oxidation during production and storage, altering their activity. This document details a method using Agilent's microfluidic-based HPLC-Chip integrated with the Accurate-Mass 6520 Q-TOF LC/MS to analyze potential oxidative modifications in monoclonal antibodies treated with hydrogen peroxide.
The study involved reducing and alkylating the antibody, followed by trypsin digestion. The resulting peptides were analyzed using MassHunter BioConfirm software. Results confirmed that peptides containing methionine and tryptophan residues were readily oxidized, allowing for the identification of specific modification sites through accurate mass measurement.
| Enrichment Column Dimensions | 40 nL |
|---|---|
| Analytical Column Dimensions | 75 mm x 43 mm |
| Flow Rate (Nanoflow LC Pump) | 600 nL/min |
| Sample Load Amount | 50 ng |
| MS Acquisition Range (MS only) | 300-3,200 m/z |
| Fragmentor Voltage | 175 V |
Peptides containing methionine and tryptophan residues were easily identified as being sensitive to oxidation.
The data were analyzed using Agilent MassHunter Qualitative Analysis software and Agilent MassHunter BioConfirm software.
A 2 uL sample of antibody (1 mg/mL) was diluted with water, and 2 uL of H2O2 (50% concentration) was added for a 20-minute reaction at room temperature.